In previous studies we have identified the chromosomal location of Unit I and Unit II endogenous MMTV genomes of BALB/c mice. We have now determined the chromosomal location of the Unit II endogenous MMTV genome using mouse/hamster somatic cell hybrids and genetic crosses between mouse strains. The Unit II MMTV genome is located on chromosome 6. Analysis of (C3H/OuJ x Czech II) x Czech II backcross mice revealed 1% recombination between the Unit II MMTV genome and the immunoglobulin kappa chain locus. The gene order with respect to other genetic loci on chromosome 6 is: centromers-Unit II MMTV/kappa chain gene-craf-cK-ras-2. Earlier studies of tumor incidence in pedigreed breeding colonies of M. cervicolor popaeus (line D) and M. musculus musculus (Czech II) demonstrated that mammary tumor development is associated with a chronic infection by an MMTV related virus. Examination of line D mammary tumor cellular DNA by restriction enzyme analysis led to the following observations: (1) each restricted tumor cellular DNA contained new MMTV related fragments that are not present in normal liver cellular DNA, (2) 53% of the pregnancy independent tumors contained a rearranged int-1 locus as compared to 7% with an altered int-2 locus, and (3) in pregnancy dependent tumors the int-1 locus was affected in 33% of the cases whereas the int-2 locus was unaffected. These results point to a correlation between the activation of the int-1 locus and pregnancy independent mammary tumors. We have previously demonstrated common host-viral junction fragments which do not correspond to either int-1 or int-2 in MMTV induced mammary tumor cellular DNA of Czech II mice. Recombinant DNA clones of one common host-viral restriction fragment has been obtained. Using unique host flanking sequences in this recombinant clone as a probe, we have been able to: (1) identify another mammary tumor with an insertion of MMTV in this region of the cellular genome, and (2) show that this new locus (designated int-3) is on chromosome 17.